Project Summary Microfluidics (lab-on-a-chip) is a promising technology for an extremely broad range of biomedical applications including drug discovery; tissue engineering; point-of-care diagnostics; cancer screening based on rare cell detection, protein, DNA, or micro-RNA biomarkers, and more recently, circulating exosomes. This proposal aims to revolutionize the biomedical microfluidic ecosystem by developing 3D printing to routinely create very small, densely integrated microfluidic devices for the biomedical sciences. Such devices are not possible with current microfluidic fabrication techniques, which typically rely on careful alignment and bonding of a handful of individually fabricated layers, each of which has a 2D component layout. In contrast, 3D printing permits all 3 dimensions of the device volume to be fully utilized for component placement and channel routing, offering the opportunity for dense component integration and small device volume (~5 mm3). For example, we include a preliminary device design for a multiplexed cell-based assay that tests both genotype (presence of the relevant gene) and phenotype (cell characteristics and/or behavior) in a device volume of only 2.2 mm 2.2 mm 1 mm. The device includes cell growth chambers, monoliths for mRNA capture and fluorescence measurement, and an integrated pump and valves. Moreover, print runs <1 hour enable fast fabrication and test cycles to dramatically speed device development. This proposal intends to initiate a virtuous circle in which 3D printed microfluidics becomes a disruptive tool for biomedical innovation, which should have a substantial impact on human health. To date, the key inhibiting factor for 3D printing has been the inability of commercial 3D printers and resins to fabricate the requisite microvoids that comprise microfluidic structures. Our group recently demonstrated that proper formulation of photosensitive resins coupled with fundamental understanding of the polymerization photophysics enables a low cost commercial stereolithographic (SL) 3D printer to fabricate voids with minimum dimensions on the order of 100 m. Aims 1 and 2 of this proposal will build on this success by developing a 3D printer and commensurate low cost resins to fabricate valves 40x smaller than what we have already demonstrated, and flow channels with cross section dimensions down to 24 x 30 m2. These advances will be used in Aim 3 to construct high density microfluidic devices that probe (1) expression of multiple genes in live cells and (2) quantify the viability of those cells in a single device. The overall objective of these studies is to develop 3D printed microfluidic systems with feature dimensions that are enabling for miniaturized cell-based bioanalysis.